Compounds for modulating mitochondrial function

ABSTRACT

Compounds and compositions that can modulate mitochondrial function in neuronal cells are provided herein, as are methods for using the compounds and compositions to treat or prevent conditions such as Alzheimer&#39;s disease. For example, compounds of Formula I, compositions containing the compounds, and methods for using the compounds and compositions are provided herein: 
                         
wherein X is absent, CH 2 , or C(O); R 1  is H, OH, CN, NO 2 , halo, C 1-3  alkyl, C 1-3  haloalkyl, C 1-3  alkoxy, C 1-3  haloalkoxy, C 3-7  cycloalkyl, amino, C 1-3  alkylamino, or di(C 1-3  alkyl)amino; R 2  is H or C 1-6  alkyl; R 3  is H, C 1-6  alkyl, —C(O)(C 1-3  alkyl), or —C(O)O(C 1-3  alkyl); R 4  is C 3-10  cycloalkyl, C 6-10  aryl, 5-10 membered heteroaryl, or 5-10 membered heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4 independently selected R 5  groups; and R 5  is OH, CN, NO 2 , halo, C 1-3  alkyl, C 1-3  haloalkyl, C 1-3  alkoxy, C 1-3  haloalkoxy, C 3-7  cycloalkyl, amino, C 1-3  alkylamino, or di(C 1-3  alkyl)amino.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/433,254, filedJun. 6, 2019, which is a divisional of U.S. Ser. No. 15/554,767, filedAug. 31, 2017 (now U.S. Pat. No. 10,336,700), which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2016/020698, having an International Filing Date of Mar. 3, 2016,which claims benefit of priority from U.S. Provisional Application No.62/127,584, filed on Mar. 3, 2015.

TECHNICAL FIELD

This invention relates to compounds that can be used to treatneurodegenerative diseases, for example, by modulating mitochondrialfunction in neuronal cells.

BACKGROUND

Neurodegenerative diseases occur when changes in the neurons of thebrain and spinal cord cause them to function abnormally, eventuallyresult in their deterioration and death. Symptoms may initially be mild,but they progressively worsen as more and more neurons die. For example,Alzheimer's disease (AD) is a devastating neurodegenerative disorderthat has no cure, and is associated with progressive cognitive declinein the aging population. Extracellular amyloid beta (Aβ) plaques andintracellular neurofibrillary tangles (NFTs) comprised ofhyperphosphorylated tau (p-tau) protein represent the major hallmarks ofAD pathology (Braak and Braak, Acta Neuropathologica, 82:239-259, 1991).The etiology of sporadic AD, which represents over 95% of all cases, isunknown, with age being the single risk factor. Familial AD (FAD) iscaused by mutations in presenilin 1 and 2 (PS1 and PS2) and amyloidprecursor protein (APP), all of which are involved in the abnormalprocessing of APP, leading to increased levels of Aβ. The specificmolecular mechanisms of sporadic and familial AD are still underinvestigation, hindering the development of effective therapeuticapproaches. Emerging data from multiple animal studies and clinicalinvestigations, however, suggest that there is a tight connectionbetween Aβ and p-tau, and development of strategies that target bothmechanisms could be beneficial (Mondragon-Rodriguez et al., Int JAlzheimers Dis, 2012:630182, 2012).

SUMMARY

Altered mitochondrial dynamics is an underlying and early event inprogression of neurodegenerative diseases such as AD, amyotrophiclateral sclerosis (ALS), Huntingon's disease (HD), and Parkinson'sdisease (PD). This document is based in part on the identification ofmolecules that can efficiently restore mitochondrial dynamics in neuronsand enhance their bioenergetics. A tricyclic pyrone compound (CP2) canrestore axonal trafficking of mitochondria in neurons, alleviating thedevelopment of behavior and memory phenotypes in multiple transgenicmouse models of FAD. As described herein, an array of new experimentalcompounds was subjected to biological screening to assess theirtoxicity, and to identify compounds that possess the ability to restoremitochondrial function in cells that express amyloid beta peptides. Fourclasses of compounds with properties superior to CP2 were identified,and several of those compounds were characterized in primary neurons.This document provides compounds from those classes, as well ascompositions containing one or more of the compounds, and methods formaking and using the compounds and compositions to treat, prevent, ordelay the onset of AD. It is to be noted that these compounds andcompositions also may be beneficial in treating, preventing, or delayingthe onset of other diseases and disorders, such as HD, PD, dementia(e.g., frontotemporal dementia), traumatic brain injury (TBI), multiplesclerosis (MS), ALS, diabetes, metabolic syndrome, cancer,chemotherapy-induced peripheral neuropathies, Down Syndrome, and aging.Such compounds also may increase health span and fecundity, promoting aprolonged period of health with increasing age, and delaying the onsetof age-related diseases.

In a first aspect, this document features a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein:

-   -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl);    -   R⁴ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁵ groups; and    -   R⁵ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.        In some embodiments, X can be CH₂. In some embodiments, wherein        R¹ can be C₁₋₃ alkyl. In some embodiments, R² can be C₁₋₆ alkyl.        In some embodiments, R³ can be H. In some embodiments, R⁴ can be        unsubstituted 5-10 membered heteroaryl. In some embodiments, R⁴        can be aryl and R⁵ can be halo or OH.

The compound of Formula (I) can be a compound of Formula (Ia):

or a pharmaceutically acceptable salt thereof.

The compound of Formula (I) can be a compound of Formula (II):

or a pharmaceutically acceptable salt thereof.wherein:

-   -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl);    -   R⁴ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁵ groups; and    -   R⁵ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.        In some embodiments, X can be CH₂. In some embodiments, R¹ can        be C₁₋₃ alkyl. In some embodiments, R² can be C₁₋₆ alkyl. In        some embodiments, R³ can be H. In some embodiments, R⁴ can be        unsubstituted 5-10 membered heteroaryl. In some embodiments, R⁴        can be aryl and R⁵ can be halo or OH.        The compound of Formula (II) can be a compound of Formula (IIa)

or a pharmaceutically acceptable salt thereof.

The compound of Formula (I) can be selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.

The compound of Formula (I) can be

or a pharmaceutically acceptable salt thereof.

In another aspect, this document features a pharmaceutical compositioncontaining a compound as provided herein, or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable carrier.

In another aspect, this document features a method for modulatingmitochondrial function in a subject. The method can includeadministering to the subject a therapeutically effective amount of acompound as provided herein, or a pharmaceutically acceptable saltthereof. The subject can be a human. The subject can be diagnosed ashaving a neurodegenerative disorder. The subject can be diagnosed ashaving diabetes, metabolic syndrome, cancer, a chemotherapy-inducedperipheral neuropathy, or Down syndrome.

In yet another aspect, this document features a method for treating aneurodegenerative disorder in a subject. The method can includeadministering to the subject a therapeutically effective amount of acompound of as described herein, or a pharmaceutically acceptable saltthereof. The method can include administering the compound in an amounteffective to reduce cognitive decline in the subject. Theneurodegenerative disorder can be AD, HD, PD, dementia, MS, or ALS. Forexample, the neurodegenerative disorder can be AD, and the method caninclude administering the compound in an amount effective to reduce theformation or amount of A plaques or neurofibrillary tangles (NFTs) inthe subject.

In still another aspect, this document features a method for reducingthe likelihood of AD in a subject at risk for developing AD. The methodcan include administering to the subject a therapeutically effectiveamount of a compound as provided herein, or a pharmaceuticallyacceptable salt thereof. The subject can be a human. The subject canhave been identified as having a mutation in a PS1, PS2, or APP genethat is associated with AD.

This document also features a method for modulating mitochondrialfunction in a cell. The method can include contacting the cell with aneffective amount of a compound as provided herein, or a pharmaceuticallyacceptable salt thereof. The amount can be effective to modulate ATPproduction, Complex I activity, NADH levels, and/or NAD⁺/NADH ratio inthe cell. The contacting can be in vitro. The amount can be effective toreduce Complex I activity. The cell can be within a subject identifiedas having hypoxia or ischemia.

In addition, this document features a method for treating cancer in asubject. The method can include administering to the subject atherapeutically effective amount of a compound as described herein, or apharmaceutically acceptable salt thereof. The cancer can be, forexample, pancreatic cancer, cervical cancer, or lymphoma. The subjectcan be a human. The method compound can be administered in an amounteffective to reduce the severity, progression, or recurrence of thecancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of the CP2 molecule. ED₅₀=0.12 μM, TD₅₀=39μM, TI=325.

FIGS. 2A and 2B are a pair of graphs plotting survival of SK-N-MC cellsafter treatment with the indicated compounds. Cells were seeded in96-well plates at 30,000 cells per well. Twenty four (24) hours later,cells were treated with 1, 2.5, 5, 10, or 20 μM of the indicatedcompounds (FIG. 2A, CP2 free base, CP2 TFA salt, and C452-C461; FIG. 2B,C462-C473). Cell viability was measured 48 hours later using an MTTassay. Cell viability is expressed as a percent of vehicle-treated(control) cells. All experiments were performed twice in triplicates.

FIGS. 3A and 3B are a pair of graphs plotting survival of primaryneurons after treatment with the indicated compounds. Primary embryoniccortical neurons were isolated as described elsewhere (Trushina et al.,Proc Natl Acad Sci USA, 100:12171-12176, 2003) and seeded on 96-wellplates at 30,000 cells per well. Seven days later, cells were treatedwith 2.5 μM and 5 μM of the indicated compounds (FIG. 3A, vehicle, CP2free base, CP2 TFA salt, and C452-C463; FIG. 3B, vehicle and C464-C473).Cell viability was measured 24 hours later with an MTT assay. Cellviability is expressed as a percent of vehicle-treated (control) cells.All experiments were performed twice in triplicates.

FIGS. 4A and 4B are a pair of graphs plotting survival of MC65 cellsafter treatment with the indicated compounds, in the presence (Tet/off)or absence (Tet/on) of Aβ expression. MC65 cells were cultured for 72hours in the presence (on) or absence (off) of tetracycline in OPTI-MEMmedia without serum. Cells (Tet/on and Tet/off) at the time of platingwere treated every 24 hours with vehicle (0.05% final DMSO) orexperimental compounds (FIG. 4A, CP2 free base, CP2 TFA salt, C452, andC453; FIG. 4B, C454-C457) at 2.5 μM and 5 μM. Cell viability wasmeasured with an MTT assay 72 hours later. Experiments were performed intriplicates. Every 96-well plate had its own on/off control. Fourexperimental compounds were tested per each plate. Experiments wererepeated independently twice.

FIGS. 5A and 5B are a pair of graphs plotting survival of MC65 cellsafter treatment with the indicated compounds, in the presence (Tet/off)or absence (Tet/on) of Aβ expression. MC65 cells were cultured for 72hours in the presence (on) or absence (off) of tetracycline in OPTI-MEMmedia without serum. Cells (Tet/on and Tet/off) at the time of platingwere treated every 24 hours with vehicle (0.05% final DMSO) orexperimental compounds (FIG. 5A, C458-C461; FIG. 5B, C462-C465) at 2.5μM and 5 μM. Cell viability was measured with an MTT assay 72 hourslater. Experiments were performed in triplicates. Every 96-well platehad its own on/off control. Four experimental compounds were tested pereach plate. Experiments were repeated independently twice.

FIG. 6A is a graph plotting survival of MC65 cells after treatment withthe indicated compounds, in the presence (Tet/off) or absence (Tet/on)of A expression. MC65 cells were cultured for 72 hours in the presence(on) or absence (off) of tetracycline in OPTI-MEM media without serum.Cells (Tet/on and Tet/off) at the time of plating were treated every 24hours with vehicle (0.05% final DMSO) or experimental compounds(C470-C473) at 2.5 μM and 5 μM. Cell viability was measured with an MTTassay 72 hours later. Experiments were performed in triplicates. Every96-well plate had its own on/off control. Four experimental compoundswere tested per each plate. Experiments were repeated independentlytwice. FIG. 6B is a series of representative images of MC65 cells after72 hours in culture under Tet/on and Tet/off conditions, demonstratingefficacy of CP2 and C460 against Aβ toxicity. Toxicity of C463 under thesame experimental conditions is shown for comparison. Cells were treatedwith 5 μM of each compound.

FIG. 7 is a graph plotting survival of Aβ-producing MC65 cells treatedwith each of the indicated compounds at 2.5 μM and 5 μM, as compared tosurvival after treatment with CP2 free base or CP2 TFA salt.

FIGS. 8A-8D show structures representing four classes of compounds thathad promising cell-protective activity in Aβ-producing MC65 cells. FIG.8A, C455; FIG. 8B, C458; FIG. 8C, C460; FIG. 8D, C472.

FIGS. 9A-9D are a series of graphs plotting the half maximal effectiveconcentration (EC₅₀) for each of four experimental compounds against Aβtoxicity in MC65 Tet on/off cells. FIG. 9A, C455; FIG. 9B, C458; FIG.9C, C460; FIG. 9D, C472.

FIG. 10 is a graph plotting the elimination rate for C₄₅₈ in brain andplasma of mice treated with 2.5 mg/kg of the compound via intrafemoralvein injection. Animals were sacrificed at 30 minutes, 1 hour, and 2hours post injection.

FIGS. 11A and 11B are graphs plotting survival of MC65 cells culturedfor 72 hours in the presence (on; FIG. 11A) or absence (off, FIG. 11B)of tetracycline in OPTI-MEM media without serum. Cells (Tet/on andTet/off) at the time of plating were treated with either vehicle (0.05%final DMSO) or experimental compounds at 2.5 μM every 24 hours. Cellviability was measured with an MTT assay 72 hours later. Experimentswere performed in triplicate, and every 96-well plate had its own on/offcontrol. Four experimental compounds were tested per plate. Experimentswere repeated independently twice.

FIGS. 12A and 12B are a pair of graphs plotting ATP levels in primaryembryonic cortical neurons that were seeded on 96-well plates at 30,000cells per well and treated with the indicated compounds atconcentrations ranging from 10 nM to 5 μM, as indicated. ATP levels weremeasured 24 hours later using an ATP determination Kit (LifeTechnologies; Carlsbad, Calif.). ATP levels are expressed as a percentof vehicle treated (control) cells. All experiments were performed twicein 6 replicates.

FIG. 13 is a graph plotting Complex I activity measured in isolatedmitochondria from mouse brain after treatment with the indicatedconcentrations of the indicated compounds or controls. Mouse brainhomogenates were prepared as described elsewhere (Nature Protocols 7(6),2012; doi:10.1038/nprot.2012.058). Isolated mitochondria (40 μg) weretreated with vehicle control (control), rotenone (Complex I inhibitor,negative control) or the indicated compound. Complex I activity wasmeasured as the decrease in absorbance at 340 nm for 5 minutes, startingthe reactions by adding ubiquinone (10 mM) to isolated mitochondria inthe reaction mixture. All experiments were performed in duplicate.

FIGS. 14A and 14B are graphs plotting NAD⁺ and NADH levels (FIG. 14A) orthe NAD⁺/NADH ratio (FIG. 14B) in primary embryonic cortical neuronsafter treatment with the indicated compounds. Neurons were seeded on96-well plates at 30,000 cells per well. Seven days later, cells weretreated with each compound at 2 μM. NAD⁺ and NADH levels were measured24 hours later using the bioluminescent NAD⁺/NADH-GLO assay (Promega;Madison, Wis.). NAD⁺ and NADH levels are expressed as a percent ofvehicle treated (control) cells (FIG. 14A). All experiments wereperformed in 6 replicates.

FIGS. 15A and 15B are graphs plotting percent survival of MC65 cellscultured in the presence of tetracycline (Tet-On) (FIG. 15A) or withouttetracycline (Tet-off) (FIG. 15B) in OPTI-MEM media without serum. Cellsat the time of plating were treated with either vehicle (0.05% finalDMSO) or CP2, C458, C764 at 100 nM, 2.5 μM, and 5 μM concentrations.Cell viability was measured with an MTT assay 72 hours after plating.Experiments were performed in triplicate, and were repeatedindependently twice.

FIG. 16 is a graph plotting Complex I activity measured in isolatedmitochondria from mouse brain homogenates. Isolated mitochondria (40 μg)were treated with vehicle (control), rotenone (Complex I inhibitor,negative control), and each of the indicated compounds at 100 nM and 5μM concentrations. Complex I activity was measured for 5 minutes as thedecrease in absorbance at 340 nm after the reaction was initiated byadding ubiquinone (10 mM) to isolated mitochondria in the reactionmixture. All experiments were performed in duplicate.

FIG. 17 is a graph plotting ATP levels in primary embryonic corticalneurons after seeding on a 96-well plate at 30,000 cells per well andbeing treated seven days later with the indicated compounds atconcentrations of 100 nM, 2 μM, and 5 μM. ATP levels were measured 24hours after treatment using the bioluminescent CellTiter-GLO 2 assay(Promega, Madison, Wis.). All experiments were performed in 6replicates.

FIGS. 18A-18C are graphs plotting the level of NAD+(FIG. 18A), the levelof NADH (FIG. 18B), and the ratio of NAD+/NADH (FIG. 18C) in primaryembryonic cortical neurons that were seeded on a 96-well plate at 30,000cells per well and treated seven days later with the indicated compoundsat concentrations 100 nM, 2 μM and 5 μM. NAD+, NADH, and NAD+/NADH ratiowere measured using a bioluminescent NAD+/NADH-GLO assay (Promega). Allexperiments were performed in 6 replicates.

FIG. 19 is a graph plotting survival of mice that were fed a high fatdiet (HFD) and untreated or treated with CP2, as indicated.

FIGS. 20A-20G are a series of graphs plotting the effect of theindicated compounds on survival of various cancer cells vs. control.FIG. 20A, HeLa cervical cancer cells; FIG. 20B, Panc1 pancreatic cancercells; FIG. 20C, P-WT: mouse embryonic fibroblasts (MEFs; control); FIG.20D, P++: MEFs; FIG. 20E, p53−/− ms T-cell lymphoma (TCL) cells; FIG.20F, Patu pancreatic cancer cells; FIG. 20G, Su pancreatic cancer cells.

FIGS. 21A-21H are a series of graphs plotting the results of doseresponse studies for CP2 in the cells used in FIGS. 20A-20G, with anadditional control. FIG. 21A, HeLa cervical cancer cells; FIG. 21B,Panc1 pancreatic cancer cells; FIG. 21C, P-WT: MEFs; control); FIG. 21D,P++: MEFs; FIG. 21E, p53−/− ms TCL; FIG. 21F, Patu pancreatic cancercells; FIG. 21G, Su pancreatic cancer cells; FIG. 21H, HPDE control.

DETAILED DESCRIPTION Definitions

For the terms “for example” and “such as,” and grammatical equivalencesthereof, the phrase “and without limitation” is understood to followunless explicitly stated otherwise. All measurements reported herein areunderstood to be modified by the term “about”, whether or not the termis explicitly used, unless explicitly stated otherwise. As used herein,the singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

The terms “treating” and “treatment” mean causing a therapeuticallybeneficial effect, such as ameliorating one or more existing symptomsand/or reducing the severity of symptoms that will or are expected todevelop.

A “therapeutically effective” amount of a compound as described hereinis typically one that is sufficient to achieve the desired effect andmay vary according to the nature and severity of the disease condition,and the potency of the compound. It will be appreciated that differentconcentrations may be employed for prophylaxis than for treatment of anactive disease.

The term “contacting” means bringing at least two moieties together,whether in an in vitro system or an in vivo system.

As used herein, “administration” refers to delivery of a compound orcomposition containing a compound provided herein by any external route,including, without limitation, IV, intramuscular, SC, intranasal,inhalation, transdermal, oral, buccal, rectal, sublingual, andparenteral administration.

The term “compound” as used herein is meant to include allstereoisomers, geometric isomers, and tautomers of the structuresdepicted. Compounds herein identified by name or structure as oneparticular tautomeric form are intended to include other tautomericforms, unless otherwise specified.

In some embodiments, a compound provided herein, or salt thereof, issubstantially isolated. By “substantially isolated” is meant that thecompound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compound providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is used herein to refer tothose compounds, materials, compositions, and/or dosage forms that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and animals (e.g., non-human mammals such asmice, rats, cats, dogs, pigs, cows, sheep, or horses) without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The term “alkyl” includes substituted or unsubstituted straight-chainalkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, etc.) and branched-chain alkyl groups (isopropyl,tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has sixor fewer carbon atoms in its backbone (e.g., C₁₋₆ for straight chain;C₃₋₆ for branched chain). The term C₁₋₆ includes alkyl groups containing1 to 6 carbon atoms. In certain embodiments, a straight chain alkyl hasthree or fewer carbon atoms in its backbone. The term C₁₋₃ includesalkyl groups containing one to three carbon atoms.

As used herein, “haloalkyl” means a hydrocarbon substituent, which is alinear or branched or cyclic alkyl, alkenyl or alkynyl substituted withone or more chloro, bromo, fluoro, or iodo atom(s). In some embodiments,a haloalkyl is a fluoroalkyl, wherein one or more of the hydrogen atomshave been substituted by fluoro. In some embodiments, haloalkyls are oneto about three carbons in length (e.g., one to about two carbons inlength, or one carbon in length).

The term “cycloalkyl” includes a substituted or unsubstituted cyclicaliphatic group which may be saturated or unsaturated. For example,cycloalkyl groups include cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. In some embodiments, cycloalkyls can havefrom three to seven carbon atoms in their ring structure; for example, acycloalkyl can have three, four, five, six, or seven carbons in the ringstructure.

The term “alkoxy” includes groups of the formula —OR, where R is analkyl as defined herein. Non-limiting examples of alkoxy groups includemethoxy, ethoxy, isopropoxy, tert-butoxy, and the like. In someembodiments, an alkoxy group can have from one to three carbons (e.g.,methyoxy, ethoxy, or propoxy).

The term “haloalkoxy” includes group of the formula —OR, where R is ahaloalkyl as defined herein. Examples of haloalkoxy groups include,without limitation, trifluoromethoxy, difluoromethoxy, etc.

“Alkylamino” includes groups of the formula —NR, where R is an alkyl asdefined herein. Non-limiting examples of alkylamino groups includemethylamino, ethylamino, isopropylamino, butylamino etc. In someembodiments, an alkylamino group can have from one to three carbons(e.g., methyoxy, ethoxy, or propoxy). The term “dialkylamino” includesgroups of the formula —NR₂, where R is an alkyl as defined herein. Insome embodiments, the alkyl groups of a dialkylamino independently canhave one to three carbons.

In general, the term “aryl” includes substituted or unsubstitutedaromatic rings, including 5- and 6-membered single-ring aromatic groups,such as benzene and phenyl. Further, the term “aryl” includesmulticyclic aryl groups, e.g., tricyclic, bicyclic, such as naphthaleneand anthracene. In some embodiments, aryls can have from six to ten(e.g., six, seven, eight, nine, or ten) ring atoms.The term “heteroaryl” means a substituted or unsubstituted mono-, bi-,tri- or polycyclic group having four to 14 ring atoms, alternativelyfive, six, nine, or ten ring atoms; having six, ten, or 14 pi electronsshared in a cyclic array; wherein at least one ring in the system isaromatic, and at least one ring in the system contains one or moreheteroatoms independently selected from the group consisting of N, O,and S. Exemplary heteroaryl groups include, for example, pyrrole, furan,thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole,pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like. Further, the term “heteroaryl” includesmulticyclic heteroaryl groups, e.g., tricyclic or bicyclic groups, suchas benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,napthyridine, indole, benzofuran, purine, benzofuran, quinazoline,deazapurine, indazole, or indolizine.

The term “heterocycloalkyl” includes substituted or unsubstitutedgroups, including but not limited to, three- to ten-membered single ormultiple rings having one to five heteroatoms, for example, piperazine,pyrrolidine, piperidine, or homopiperazine. In certain embodiments, aheterocycloalkyl can have from four to ten ring atoms. The term“substituted” means that an atom or group of atoms replaces hydrogen asa “substituent” attached to another group. For aryl and heteroarylgroups, the term “substituted”, unless otherwise indicated, refers toany level of substitution, namely mono, di, tri, tetra, or pentasubstitution, where such substitution is permitted. The substituents areindependently selected, and substitution may be at any chemicallyaccessible position. In some cases, two sites of substitution may cometogether to form a 3-10 membered cycloalkyl or heterocycloalkyl ring.Non-limiting examples of substituents include: (C₁-C₆)alkyl, halo,(C₁-C₆)haloalkyl, —CN, —NR⁸R⁹, —NO₂, —O(C₁-C₆)haloalkyl, —OR⁸, —OC(O)R⁸,—C(O)R⁸, —C(O)OR⁸, —C(O)NR⁸R⁹, —SR⁸, —S(O)R⁸, —SO₂R⁸, —SO₂NR⁸R⁹, (C₃-C₇)cycloalkyl, (C₃-C₇)heterocycloalkyl, (C₅-C₁₄)aryl, and(C₅-C₁₄)heteroaryl, wherein R⁸ and R⁹ are independently selected from Hand (C₁-C₆)alkyl.

Modulators of Mitochondrial Function

This document provides compounds that can modulate mitochondrialfunction and induce metabolic reprogramming, as well as methods andmaterials for using such compounds to treat disorders such as AD andother neurological conditions. Thus, in some embodiments, this documentprovides compounds that can be used to restore mitochondrial function incells (e.g., primary neurons) that express Aβ peptides. Provided hereinare, inter alia, the following compounds:

or a pharmaceutically acceptable salt thereof.

It is to be noted that this document encompasses not only the variousisomers, diastereomers, enantiomers, and tautomers that may exist, butalso the various mixtures of isomers, diastereomers, enantiomers, andtautomers that may be formed.

The scope of this document also encompasses solvates and salts of thecompounds described herein, as well as prodrugs of the compounds, suchas esters, amides, and acylated groups, among others. In someembodiments, for example, this document provides prodrugs of thecompounds disclosed herein, which may contain, for example, acylatedphenols or acyl derivatives of amines. By “prodrug” is meant, forexample, any compound (whether itself active or inactive) that isconverted chemically in vivo into a biologically active compound asprovided herein, following administration of the prodrug to a subject.In some embodiments, a prodrug is a covalently bonded carrier thatreleases the active parent drug when administered to a subject. Prodrugscan be prepared by modifying functional groups present in the compoundsin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compounds. Prodrugs can includecompounds in which hydroxyl, amino, sulfhydryl, or carboxyl groups arebonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl grouprespectively. Examples of prodrugs include, without limitation, acetate,formate and benzoate derivatives of alcohol and amine functional groupsin the compounds provided herein. The suitability and techniquesinvolved in making and using prodrugs are discussed in Higuchi andStella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the ACSSymposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are hereby incorporated by reference in theirentirety.

Also provided herein is a compound of Formula (I):

or a pharmaceutically acceptable salt thereof,where:

-   -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl);    -   R⁴ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁵ groups; and    -   R⁵ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, X is CH₂. In some embodiments, R¹ is C₁₋₃ alkyl.For example, R¹ can be methyl. In some embodiments, R² is C₁₋₆ alkyl.For example, R² can be methyl. In some embodiments, R³ is H. In someembodiments, R⁴ is unsubstituted 5-10 membered heteroaryl. For example,R⁴ can be phenyl, pyridinyl, cyclohexanyl, and benzoimidazolyl. In someembodiments, R⁴ is aryl (e.g., phenyl) and R⁵ is halo (e.g., chloro) orOH.

In some embodiments, the compound of Formula (I) can have the followingstereochemistry:

where R¹, R², R³, and R⁴ are as set forth above.In some embodiments, the compound of Formula (I) can be a compound ofFormula (II):

or a pharmaceutically acceptable salt thereof,where:

-   -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl);    -   R⁴ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁵ groups; and    -   R⁵ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

In some embodiments, R¹ is C₁₋₃ alkyl. For example, R¹ can be methyl. Insome embodiments, R² is C₁₋₆ alkyl. For example, R² can be methyl. Insome embodiments, R³ is H. In some embodiments, R⁴ is unsubstituted 5-10membered heteroaryl. For example, R⁴ can be phenyl, pyridinyl,cyclohexanyl, and benzoimidazolyl. In some embodiments, R⁴ is aryl(e.g., phenyl) and R⁵ is halo (e.g., chloro) or OH.

In some embodiments, the compound of Formula (II) can have the followingstereochemistry:

where R¹, R², R³, and R⁴ are as set forth above.

Also provided herein is a compound of Formula (III):

or a pharmaceutically acceptable salt thereof,where:

-   -   V, W, Y, and Z are independently selected from the group        consisting of CH and N;    -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   m is an integer from 0 to 2;    -   n is an integer from 0 to 2;    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H and C₁₋₆ alkyl;    -   R⁴ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and C(O)O(C₁₋₃ alkyl);    -   R⁵ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁶ groups; and    -   R⁶ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

As indicated in Formula (III), the central cycloalkyl ring can be acyclopentyl, cyclohexyl, or cycloheptyl ring. It is to be noted that insome embodiments, the central ring may be a phenyl ring or aheterocyclic structure (e.g., a pyridyl ring).

Thus, in some embodiments, this document also provides compounds ofFormulae (IV)-(VII):

or a pharmaceutically acceptable salt thereof,where:

-   -   the central aromatic ring contains 1 or more nitrogen atoms;    -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl);    -   R⁴ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁵ groups; and    -   R⁵ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

Also provided herein is a compound of Formula (VIII):

or a pharmaceutically acceptable salt thereof,where:

-   -   A, B, D, E, W, Y, and Z are independently selected from the        group consisting of CH and N;    -   X is absent or selected from the group consisting of CH₂ and        C(O);    -   n is an integer from 0 to 2;    -   R¹ is selected from the group consisting of H, OH, CN, NO₂,        halo, C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy,        C₃₋₇ cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃        alkyl)amino;    -   R² is selected from the group consisting of H and C₁₋₆ alkyl;    -   R³ is selected from the group consisting of H and C₁₋₆ alkyl;    -   R⁴ is selected from the group consisting of H, C₁₋₆ alkyl,        —C(O)(C₁₋₃ alkyl), and C(O)O(C₁₋₃ alkyl);    -   R⁵ is selected from the group consisting of C₃₋₁₀ cycloalkyl,        C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered        heterocycloalkyl, each optionally substituted by 1, 2, 3, or 4        independently selected R⁶ groups; and    -   R⁶ is selected from the group consisting of OH, CN, NO₂, halo,        C₁₋₃ alkyl, C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇        cycloalkyl, amino, C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.

Again, it is to be noted that this document encompasses not only thevarious isomers, diastereomers, enantiomers, and tautomers that mayexist for the compounds described herein, but also the various mixturesof isomers, diastereomers, enantiomers, and tautomers that may be formed

Non-limiting examples of compounds according to the above Formulaeinclude:

or a pharmaceutically acceptable salt thereof.

Most of these compounds also are shown in TABLE 3 below. Methods formaking such compounds include those known in the art and describedherein.

In some embodiments, these compounds can have the followingstereochemistry:

As used herein, chemical structures that contain one or morestereocenters depicted with bold and dashed bonds are meant to indicateabsolute stereochemistry of the stereocenter(s) present in the chemicalstructure. As used herein, bonds symbolized by a simple line do notindicate a stereo-preference. Unless indicated to the contrary, chemicalstructures that include one or more stereocenters and are illustratedherein without indicating absolute or relative stereochemistry encompassall possible steroisomeric forms of the compound (e.g., diastereomersand enantiomers) and mixtures thereof. Structures with a single bold ordashed line, and at least one additional simple line, encompass a singleenantiomeric series of all possible diastereomers.

A compound provided herein, including a pharmaceutically acceptable saltthereof, can be purchased commercially or prepared using organicsynthesis techniques. See, for example, the Examples herein.

A reaction for preparing a compound provided herein can be carried outin suitable solvents that can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of a compound can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups, can be readilydetermined by one skilled in the art. The chemistry of protecting groupscan be found, for example, in Protecting Group Chemistry, 1st Ed.,Oxford University Press, 2000; and March's Advanced Organic chemistry:Reactions, Mechanisms, and Structure, 5th Ed., Wiley-IntersciencePublication, 2001 (each of which is incorporated herein by reference inits entirety).

Pharmaceutically Acceptable Salts and Compositions

This document also provides pharmaceutically acceptable salts of thecompounds provided herein. Examples of pharmaceutically acceptable saltsof the compounds provided herein include acid addition salts and basesalts of the compounds.

Suitable acid addition salts are formed from acids that form non-toxicsalts. Examples include the acetate, adipate, aspartate, benzoate,besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate,citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate,gluconate, glucuronate, hexafluorophosphate, hibenzate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate,malonate, mesylate, methylsulphate, 2-napsylate, nicotinate, nitrate,orotate, oxalate, palmitate, pamoate, phosphate/hydrogen,phosphate/phosphate dihydrogen, pyroglutamate, saccharate, stearate,succinate, tannate, D- and L-tartrate, 1-hydroxy-2-naphthoate tosylate,and xinafoate salts.

Suitable base salts are formed from bases that form non-toxic salts.Examples include the aluminium, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine, and zinc salts.

Hemisalts (e.g., hemisulphate and hemicalcium salts) of acids and basesmay also be formed.

A compound provided herein intended for pharmaceutical use can beadministered as a crystalline or amorphous product. In some cases, sucha product can be obtained, for example, as a solid plug, powder, or filmby methods such as precipitation, crystallization, freeze drying, spraydrying, or evaporative drying. Microwave or radio frequency drying maybe used for this purpose.

A compound can be formulated for administration by any route, includingorally, rectally, sublingually, and parenterally. Parenteraladministration includes, for example, intravenous, intramuscular,intraarterial, intraperitoneal, intranasal, intravaginal, intravesical(e.g., to the bladder), intradermal, transdermal, topical orsubcutaneous administration. Also contemplated is the installation of acompound in the body of a patient in a controlled formulation, withsystemic or local release of a compound to occur at a later time. Forexample, a compound can be localized in a depot for controlled releaseto the circulation, or for release to a local site. Advantageously, acompound can be administered in the form of a pharmaceuticalcomposition.

This document also provides pharmaceutical compositions containing oneor more compounds as described herein, or an individual isomer, racemicor non-racemic mixture of isomers, or a pharmaceutically acceptable saltor solvate thereof, together with at least one pharmaceuticallyacceptable carrier, and optionally other therapeutic and/or prophylacticingredients.

A compound as provided herein can be administered alone or incombination with one or more other compounds provided herein, or incombination with one or more other drugs (or as any combinationthereof). Generally, a compound will be administered as a formulation inassociation with one or more pharmaceutically acceptable excipients. Theterm “excipient” is used herein to describe any ingredient other than acompound(s) provided herein. The choice of excipient will to a largeextent depend on factors such as the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form.

Non-limiting examples of pharmaceutical excipients suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. Pharmaceutically acceptableexcipients include, without limitation, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate,sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethyl cellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of a compound providedherein. In some embodiments, the excipient is a physiologicallyacceptable saline solution.

In some embodiments, a pharmaceutical composition can be formulated intosuitable pharmaceutical preparations such as solutions, suspensions,tablets, dispersible tablets, pills, capsules, powders, sustainedrelease formulations or elixirs, for oral administration or in sterilesolutions or suspensions for parenteral administration, as well astransdermal ointments, creams, gels, and patch preparations and drypowder inhalers (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition 1985, 126).

The concentration of a compound in a pharmaceutical composition willdepend on absorption, inactivation, and excretion rates of the compound,the physicochemical characteristics of the compound, the dosageschedule, and the amount administered, as well as other factors known tothose of skill in the art.

A pharmaceutical composition can be administered at once, or can bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular patient, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

The pharmaceutical compositions can be provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compound(s). The pharmaceuticallytherapeutically active compounds are, in some embodiments, formulatedand administered in unit-dosage forms or multiple-dosage forms.Unit-dose forms as used herein refers to physically discrete unitssuitable for human and animal patients and packaged individually as isknown in the art. Each unit-dose contains a predetermined quantity ofthe therapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses that are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can be prepared by,for example, dissolving, dispersing, or otherwise mixing a compound asprovided herein and optional pharmaceutical adjuvants in a carrier suchas, without limitation, water, saline, aqueous dextrose, glycerol,glycols, ethanol, and the like, to form a solution or suspension. Ifdesired, a pharmaceutical composition to be administered also cancontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, solubilizing agents, pH buffering agents andthe like (e.g., acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents).

Dosage forms or compositions can contain a compound as provided hereinin the range of 0.005% to 100%, with the balance made up from one ormore non-toxic carriers. Methods for preparation of these compositionsare known to those skilled in the art. The contemplated compositions maycontain 0.001%-100% active ingredient. In some embodiments, for example,a composition can contain 0.1-95% active ingredient, and in otherembodiments, a composition can contain 75-85% active ingredient.

Pharmaceutical compositions suitable for the delivery of compoundsprovided herein, as well as methods for their preparation, will bereadily apparent to those skilled in the art. Such compositions andmethods for their preparation can be found, for example, in Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

Methods of Use

This document also provides methods and materials for using compoundsthat modulate (e.g., increase or decrease) the activity of mitochondrialComplex I and affect cellular energetics. Mitochondrial bioenergeticsinclude the production of energy from nutrients in the form of glucoseand fat, and are conducted in the mitochondrial matrix using enzymaticcomplexes of oxidative phosphorylation machinery (e.g., Complex I).Thus, via modulation of Complex I activity, cellular energetics as awhole can be modulated. For example, a compound as provided herein candecrease the activity of Complex I that results in decreased basalrespiration of mitochondria, thus affecting bioenergetics. It is to benoted that decreasing the activity of Complex I also may improverecovery from ischemia or reperfusion, which can result in inflammationand oxidative damage through the induction of oxidative stress as bloodreturns to the tissue after a period of ischemia or lack of oxygen.Thus, the compounds and compositions described herein can providecardioprotection from hypoxia/ischemia, and can be used to treatsubjects identified as being in need of such cardioprotection.

In some cases, a compound as provided herein can be used to treat adisease or disorder that involves mitochondrial dysfunction. Forexample, mitochondrial motility can be restored in a patient byadministering a therapeutically effective amount of a compound providedherein. In addition, mitochondrial function can be increased in a cellby contacting the cell with an effective amount of a compound providedherein.

Diseases and disorders that involve mitochondrial dysfunction include,for example, neurodegenerative diseases such as AD, HD, PD, varioustypes of dementia (e.g., frontotemporal dementia), MS, amyotrophiclateral sclerosis (ALS), diabetes, metabolic syndrome, cancer,chemotherapy-induced peripheral neuropathies, and Down Syndrome, as wellas aging. In some embodiments, the compounds provided herein also canpromote healthy aging, and increase longevity and fecundity.

Compounds provided herein are effective to modulate mitochondrialfunction in a cell, for example, in a neural cell. Therefore, thisdocument also provides a method of modulating (e.g., restoring)mitochondrial dynamics in a cell, comprising contacting the cell with aneffective amount of a compound provided herein, or a pharmaceuticallyacceptable salt form thereof. The method can be performed by contactingthe cell with a compound as described herein, or a pharmaceuticallyacceptable salt form thereof, in vitro, thereby modulating mitochondrialdynamics and function in vitro. Uses of such an in vitro method include,without limitation, use in a screening assay (e.g., wherein a compounddescribed herein is used as a positive control or standard compared tocompounds of unknown activity or potency in modulating mitochondrialfunction).

In the methods provided herein, any appropriate method can be used toadminister a compound to a subject. Administration can be, for example,parenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous drip).Administration can be rapid (e.g., by injection) or can occur over aperiod of time (e.g., by slow infusion or administration of slow releaseformulations). In some embodiments, administration can be topical (e.g.,transdermal, sublingual, ophthalmic, or intranasal), pulmonary (e.g., byinhalation or insufflation of powders or aerosols), or oral.

A compound provided herein can be administered to a subject in anappropriate amount, at an appropriate frequency, and for an appropriateduration effective to achieve a desired outcome (e.g., to reduce one ormore clinical symptoms or molecular/cellular hallmarks of aneurodegenerative disease such as AD, to protect or restoremitochondrial function in cells within a subject with the disease, or toreduce the likelihood of the disease or delay or prevent the onset ofthe disease in a subject at risk of developing the disease, such as asubject carrying a mutation in a PS1, PS2, or APP gene that isassociated with AD, or carrying an ApoE4 allele that predisposes asubject to the development of AD). When the disease is AD, symptoms andhallmarks that can be reduced by treatment with a compound orcomposition as provided herein include, for example, cognitive decline,formation of extracellular A plaques and/or intracellular NFTs, andabnormal processing of APP. Thus, administration of an effective amountof a compound or composition as provided herein can result in improvedcognitive function, reduced formation or numbers of Aβ plaques and/orNFTs (e.g., reduced by at least about 5 percent, about 10 percent, about25 percent, about 50 percent, about 75 percent, about 90 percent, ormore than 90 percent, as compared to the formation or number of Aplaques and/or NFTs in the subject prior to administration of thecompound or composition, or in a control subject or population ofsubjects to whom the compound or composition was not administered),and/or reduced abnormal processing of APP in a subject. Any suitablemethod can be used to assess cognitive function/decline, Aβ plaqueformation or number, NFT formation or number, and APP processing in asubject. Such methods can include, without limitation,neuropsychological tests (e.g., question-and-answer tests or othertasks) that measure memory, language skills, ability to do arithmetic,and other abilities related to brain functioning; tests of blood and/orcerebrospinal fluid, or other medical tests to assess neurologicalfunctioning associated with dementia; computed tomography (CT) scan ormagnetic resonance imaging (MRI) tests that can reveal changes in brainstructure that indicate AD; other imaging techniques, such as positronemission tomography (PET) and single photon emission computed tomography(SPECT); PIB-PET imaging that can detect amyloid in the brain of aliving patient; and emerging methods of diagnosis such as metabolomicand epigenetic profiling.

Further, administration of an effective amount of a compound orcomposition as provided herein can result in enhanced ability to sustainoxidative damage or produce energy under stress conditions, and/or inrestored mitochondrial dynamics in cells of a subject, where themitochondrial function is increased by at least about 5% (e.g., about25%, about 50%, about 75%, or more than about 75%). Mitochondrialfunction in cells of a subject can be assessed by, for example,evaluating levels of spare respiratory capacity, axonal motility, ATPproduction, coupling efficiency of respiratory chain, level of protonleak, Complex I activity, and/or NAD⁺/NADH ratios, using methods such asthose described herein. In some embodiments, mitochondrial function canbe assessed in peripheral cells (e.g., fibroblasts or lymphocytes) usingmethods described herein, including evaluation of Complex I-V activity,ATP production, and resistance to oxidative damage and stress.Parameters of mitochondrial energetics in cells from treated AD patientsalso can be evaluated using, e.g., a Seahorse Extracellular FluxAnalyzer (see, Lange et al., Frontiers Neurol, 3:175, 2012).Mitochondrial function in AD patients also can be assessed using FDG-PETto monitor levels of glucose utilization in affected brain regions, andby measuring levels of mitochondrial metabolites using proton magneticresonance (MR) spectroscopy such as lactate, N-acetyl aspartate (NAA),and mio-inositol. In addition, mitochondrial function can be assessed inblood, plasma, CSF, and/or peripheral cells/tissue by application ofmetabolomics profiling (see, e.g., Trushina et al., PLOS ONE, 8:e63644,2013).

Optimum dosages of a compound or composition as provided herein can varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC₅₀ found to be effective in in vitroand in vivo animal models. Dosages may fall within the range from 0.5 mgto 500 mg. For example, an effective amount of a compound as providedherein can be from about 0.5 mg to about 1 mg, about 1 mg to about 5 mg,about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg toabout 50 mg, about 50 to about 100 mg, about 100 mg to about 250 mg, orabout 250 mg to about 500 mg. If a particular subject fails to respondto a particular amount, then the amount of the compound or compositioncan be increased by, for example, two fold. After receiving this higherconcentration, the subject can be monitored for both responsiveness tothe treatment and toxicity symptoms, and adjustments made accordingly.The effective amount can remain constant or can be adjusted as a slidingscale or variable dose depending on the subject's response to treatment.Various factors can influence the actual effective amount used for aparticular application. For example, the frequency of administration,duration of treatment, and severity of disease may require an increaseor decrease in the actual effective amount administered.

The frequency of administration can be any frequency that has a desiredeffect (e.g., reducing one or more clinical symptoms ormolecular/cellular hallmarks of AD, increasing or restoringmitochondrial function in cells within a subject with AD, or delaying orpreventing the onset of AD in a subject at risk of AD), withoutproducing significant toxicity. For example, the frequency ofadministration can be once or more daily, biweekly, weekly, monthly, oreven less. The frequency of administration can remain constant or can bevariable during the duration of treatment. A course of treatment caninclude rest periods. For example, a composition containing one or morecompounds as provided herein can be administered over a two week periodfollowed by a two week rest period, and such a regimen can be repeatedmultiple times. As with the effective amount, various factors caninfluence the actual frequency of administration used for a particularapplication. For example, the effective amount, duration of treatment,route of administration, and severity of disease may require an increaseor decrease in administration frequency.

An effective duration for administering a compound provided herein canbe any duration that has a desired effect (e.g., for AD, reducing one ormore clinical symptoms or molecular/cellular hallmarks of AD, enhancingor restoring mitochondrial function in cells within a subject with AD,or delaying or preventing the onset of AD in a subject at risk of AD),without producing significant toxicity. Thus, an effective duration canvary from several days to several weeks, months, or years, but ingeneral, an effective duration for treatment of AD can extend for numberof years, such that an effective duration can be for as long as anindividual subject is alive. Multiple factors can influence the actualeffective duration used for a particular treatment. For example, aneffective duration can vary with the frequency of administration,effective amount, use of multiple treatment agents, route ofadministration, and severity of the disease.

After administering a compound as provided herein to subject having orat risk for developing a disorder such as AD, the subject can bemonitored to determine whether or not the disorder has been treated orprevented. For AD, for example, a subject can be assessed aftertreatment to determine whether or not a symptom of AD has been reduced,or whether any symptoms or hallmarks of AD have developed (e.g., in thecase of a person at risk for developing AD). Methods for assessingsymptoms and hallmarks of AD are described herein.

In some embodiments, an effective amount of a compound as describedherein, or a composition containing a compound as described herein, canbe any amount that reduces the severity, progression, or recurrence ofcancer (e.g., pancreatic cancer, cervical cancer, or lymphoma) in asubject (e.g., a human or a non-human mammal), without producingsignificant toxicity to the subject. Effective doses can vary dependingon the severity of the cancer, the route of administration, the age andgeneral health condition of the subject, excipient usage, thepossibility of co-usage with other therapeutic treatments, and thejudgment of the treating physician. If a recipient fails to respond to aparticular amount, then the amount of a compound or composition can beincreased by, for example, two fold. After receiving this higher amount,the subject can be monitored for both responsiveness to the treatmentand toxicity symptoms, and adjustments made accordingly. The effectiveamount can remain constant or can be adjusted as a sliding scale orvariable dose depending on the response to treatment. Various factorscan influence the actual effective amount used for a particularapplication. For example, the frequency of administration, duration oftreatment, use of multiple treatment agents, route of administration,and severity of the cancer may require an increase or decrease in theactual effective amount administered.

The frequency of administration can be any frequency that reduces theseverity, progression, or recurrence of cancer (e.g., pancreatic cancer,cervical cancer, or lymphoma) in a subject (e.g., a human or a non-humanmammal) without producing significant toxicity to the subject. Forexample, the frequency of administration can be from about once a weekto about three times a day. The frequency of administration can remainconstant or can be variable during the duration of treatment. A courseof treatment with a composition containing one or more of the compoundsdescribed herein can include rest periods. For example, a compositioncan be administered daily over a two week period followed by a two weekrest period, and such a regimen can be repeated multiple times. As withthe effective amount, various factors can influence the actual frequencyof administration used for a particular application. For example, theeffective amount, duration of treatment, use of multiple treatmentagents, route of administration, and severity of the cancer may requirean increase or decrease in administration frequency.

An effective duration for administering a composition containing one ormore compounds as described herein can be any duration that reduces theseverity, progression, or recurrence of cancer (e.g., pancreatic cancer,cervical cancer, or lymphoma) without producing significant toxicity tothe recipient. Thus, the effective duration can vary from several daysto several weeks, months, or years. In general, the effective durationfor the treatment with one or more compounds as described herein toreduce the severity, progression, or recurrence of cancer (e.g.,pancreatic cancer, cervical cancer, or lymphoma) can range in durationfrom one week to one year (e.g., one month to six months). Multiplefactors can influence the actual effective duration used for aparticular treatment. For example, an effective duration can vary withthe frequency of administration, effective amount, use of multipletreatment agents, route of administration, and severity of the cancerbeing treated.

In some embodiments, a course of treatment and the severity of one ormore symptoms related to the cancer being treated (e.g., pancreaticcancer, cervical cancer, or lymphoma) can be monitored. Any appropriatemethod can be used to determine whether or not the severity of a symptomis reduced. For example, the severity of a symptom of pancreatic cancer(e.g., cancer recurrence) can be assessed using imaging techniques.

Also provided herein are articles of manufacture containing one or morecompounds as described herein, or a pharmaceutical compositioncontaining one or more compounds as described herein, in combinationwith a pharmaceutically acceptable carrier, for example. The compound orcomposition can be within a container (e.g., a bottle, vial, orsyringe). The article of manufacture also can include a label withdirections for reconstituting and/or using the compound(s) orcomposition. In some embodiments, an article of manufacture can includeone or more additional items (e.g., one or more buffers, diluents,filters, needles, syringes, and/or package inserts with furtherinstructions for use).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLE Example 1—Identification of Small Molecule Compounds forRestoring Mitochondrial Function

An array of 24 compounds was synthesized and purified. CP2 (FIG. 1), atricyclic pyrone compound that protects against amyloid and mutanthuntingtin toxicity in cellular and animals models of AD and HD(Trushina et al., BMC Neurosci, 10:73, 2009; Rana et al., Bioorg MedChem Lett, 19:670-674, 2009; Maezawa et al., J Neurochem, 98:57-67,2006; Hong et al., J Neurochem, 108(4):1097-1108, 2009 (published online25 Dec. 2008); and Zhang et al., eBioMedicine, 2015), was synthesized asa TFA salt and free base to provide a reference compound for biologicaltesting. All synthesized compounds were HPLC purified and had a finalpurity over 95%.

All 24 compounds were screened to test their toxicity and efficacy.Specifically, the toxicity of the compounds was evaluated in (1) theSK-N-MC cell line, a parental cell line for the Tet on/off MC65 cells,and (2) primary mouse cortical neurons. In addition, the efficiency ofeach compound (along with CP2) was evaluated against Aβ toxicity in MC65cells, Tet on/off cells generated to express Aβ.

Example 2—Toxicity in SK-N-MC Neuroepithelioma Cells

Toxicity of the 24 new compounds, CP2 free base, and CP2 TFA salt wasevaluated in the SK-N-MC neuroepithelioma cell line. SK-N-MC is aparental cell line for MC65 cells that conditionally express A and havebeen used previously to test the effect of CP2 against Aβ toxicity(Maezawa et al., J Neurochem, 98:57-67, 2006). SK-N-MC were treated at 1μM, 2.5 μM, 5 μM, 10 μM and 20 μM with each compound or vehicle (DMSO0.05%-water). Cell viability was measured using a3-(4,5-dimethylthiazole-2-yl)-2,5-dyphenyltetrazolium bromide (MTTassay) after 48 hours. As shown in FIGS. 2A and 2B, most of thecompounds did not cause toxicity at 1-20 μM, except for compounds 463and 464, where treatment with 10 μM resulted in 50% cell death.Compounds 452-454, 456-462, 470, and 471 became modestly toxic (20%) inthe range of 2.5 μM to 5 μM. Thus, this range of concentrations wasselected for the rest of the screening studies.

Example 3—Toxicity in Primary Mouse Cortical Neurons

Toxicity of the 24 compounds, CP2 free base, and CP2 TFA salt wasevaluated in primary mouse embryonic (E17) cortical neurons (FIGS. 3Aand 3B). Most of the compounds were not toxic or had low (˜20%) toxicity(C453, C457, C461) at concentrations of 2.5 μM to 5 μM.

Example 4—Efficacy of Novel Compounds Against Aβ Toxicity in MC65 CellLine

The efficacy of all 24 compounds against Aβ toxicity was evaluated inMC65 cells. These cells are stably transfected with A expression vectorunder tetracycline control, and were used to study CP2 propertiesagainst Aβ toxicity as described elsewhere (Maezawa et al., J Neurochem,98:57-67, 2006). The viability of MC65 cells expressing Aβ (Tet/off, 40%viability) or not expressing Aβ (Tet/on, 100% viability) was measuredafter treatment with individual compounds. Results for all 24 compoundsagainst A toxicity are presented in FIGS. 4A, 4B, 5A, 5B, 6A, and 6B.

The efficacy of all 24 compounds against Aβ toxicity was compared tothat of CP2 free base and CP2 TFA salt (FIG. 7). These studies led tothe identification of four compounds (FIGS. 8A-8D; C455, C458, C460, andC472, respectively) that had promising cell protective activity inAβ-producing MC65 cells. Various properties of these four compounds arepresented in TABLE 1. The four compounds were selected for furtherstudies to assess mitochondrial function and dynamics in neurons.

TABLE 1 Drug-like properties of hit molecules C455 C458 C460 C472Calculated log P 3.1 4.4 0.81 0.3 Calculated log D_(7.4) 2.0 2.0 0.2 0.3Total polar surface area 62.9 34.2 92.0 118.3 H-bond acceptors 5 3 7 9H-bond donors 1 1 2 2 Molecular weight 367.45 338.50 382.42 341.90

Example 5—Measurement of EC₅₀ for Four Selected Compounds

To further characterize the efficacy of these four compounds, the halfmaximal effective concentration (EC₅₀) against Aβ toxicity in MC65 cellswas measured using an MTT assay. EC₅₀ values were calculated by fittingexperimental data to calculated data for nonlinear regression usingGraphPad Prizm5 software. The results are presented in FIGS. 9A-9D.Compound C458 demonstrated 100-fold higher efficacy than C455 and C460,and 20- to 30-fold higher efficacy than CP2, with EC₅₀ near 4 nM.

Example 6—Toxicity, Histology, and Pharmacokinetics (PK) of C458

The feasibility of developing the C458 series was evaluated bydetermining the in vivo PK profile for C458. Re-synthesized C458 wasadministered intravenously to wild type mice at 2 mg/kg. Frozen serumand brain tissue samples were analyzed using a bioanalytical LC-MS/MSmethod. The resulting PK data showed that the compound persisted in theblood with an apparent t/2 of 48 min (TABLE 2). Further, the compoundshowed significant CNS penetration, as evidenced concentrations in thebrain tissue that were 7-10 times higher (w/v) than in plasma.

Treatment of wild type (WT) mice with C458 (50 mg/kg for one month adlibitum in drinking water) did not cause histological abnormalities inanimals. As noted above, C458 demonstrated the highest efficacy againstAβ toxicity in an MC65 cell-based assay, with EC₅₀=3.59 nM (30-foldlower than CP2, which had an EC₅₀=120 nM). To evaluate the toxicity ofC458 in vivo, 2-month old breeding WT mice were treated with 50 mg/kg ofC458 via drinking water. Untreated littermates were used as controls.After one month of treatment, adult and newborn animals were sacrificedand tissue was collected for histological examination. Histology resultsdemonstrated lack of any developmental or tissue pathology, as organs ofC458-treated mice were indistinguishable from those of untreatedanimals. Similarly, no pathological abnormalities were detected innewborn mice conceived by breeding WT mice treated with C458 (50 mg/kgfor one month ad libitum in drinking water).

Example 7—Elimination Rate and Half-Life of C458 in Plasma and BrainTissue

The elimination rate and half-life of C458, measured in plasma andbrain, demonstrated good bioavailability of C458, and again suggestedthat C458 penetrates the blood brain barrier. A rough estimation ofelimination rate and half-life of C458 was determined in vivo bydelivering a dose of 2.5 mg/kg to each mouse via intrafemoral veininjection. Each group of animals (n=2) was sacrificed at 30 minutes, 1hour, and 2 hours post injection. Blood and brain tissue was collected,flash frozen, and the concentration of C458 was estimated using aLC-MS/MS assay with a linear calibration curve from 1 ng/ml to 1 ug/mlin both plasma and brain. The concentration of C458 in the brain was 7-8times higher than in plasma, suggesting that C458 penetrates the bloodbrain barrier and accumulates in the brain.

For rough estimation of elimination rate and half-life, aone-compartment model and the first rate elimination was assumed.Plotting (Ln) of concentration vs. time provided the elimination rateand half-life of C458 in plasma and the brain (FIG. 10). The estimatedhalf-life of C458 for the first rate elimination (defined as t_(1/2)=ln2/k) in plasma and brain was 48 minutes and 42 minutes, respectively.

TABLE 2 PK data for C458 Sample Mouse Time Plasma Concentration BrainConcentration ID ID (hr.) (ng/mL) (ng/g) 0001 WT 116 0.5 43.7 316 0002WT121 0.5 41.4 390 0003 WT 130 1 15.2 149 0004 WT 131 1 13.0 118 0005 WT145 2 9.83 68.2 0006 WT 156 2 10.3 76.4

Example 8—Expansion of the C458 Series

An array of 23 compounds as synthesized and purified (TABLE 3). CompoundC458 was re-synthesized as both the TFA and HCl salt to provide areference compound. All synthesized compounds were HPLC purified and hada final purity over 95%.

TABLE 3 C458 series compounds MW % (free Structure Purity base) FW(salt)salt Comments

99.5 338.50 452.52 TFA Highly hygroscopic

100.0 338.50 374.96 HCl HCl form of control compound

76.9 351.54 388.00 HCl Control compound for PK

99.9 338.50 374.96 HCl Sent for PK

100.0 338.50 374.96 HCl

97.6 324.47 360.93 HCl One less CH₂

100.0 343.56 380.02 HCl Non- aromatic

96.7 338.50 338.50 HCl 2-pyridyl isomer

100.0 371.95 371.95 HCl

98.5 352.52 388.98 HCl

99.7 338.50 374.96 HCl

100.0 371.95 408.41 HCl

98.1 352.52 388.98 HCl

100.0 371.95 408.41 HCl

98.6 385.98 422.44 HCl

100.0 353.51 389.97 HCl

98.6 352.52 388.98 HCl Aniline version

99.1 353.51 389.97 HCl

98.8 353.51 389.97 HCl

97.8 339.48 375.94 HCl Adjust pKB

100.0 380.53 416.99 HCl

96.5 352.48 388.94 HCl Non-basic N

99.0 377.53 413.99 HCl Fused ring

Example 9—Preparation of StartingMaterial—cis-(2-(3-(m-tolyloxy)cyclohexyl)propan-1-ol)

Step 1: Preparation of prop-1-en-2-ylmagnesium Bromide

Magnesium turnings (6.2 g, 252.6 mmol, 1.05 equiv.) were suspended indry tetrahydrofuran (500 mL) in a one liter flask fitted with a magneticstirring bar and an N₂ inlet. Isopropenyl bromide (5 mL, 57.2 mmol, 0.24equiv.) and a small iodine crystal (˜1 mm dia.) were added and themixture was heated to 60° C. The reaction initiated in about fifteenminutes: the heating bath was then turned off and more isopropenylbromide (16 mL, 182.9 mmol, 0.76 equiv.) was added in 4 mL portions. Thevigorous reaction was allowed to subside in between additions. Theresulting pale yellow solution of prop-1-en-2-ylmagnesium bromide wasallowed to cool to room temperature and used in the next step. Thetheoretical concentration of the solution is 0.48M. Only a trace ofundissolved magnesium remained.

Step 2—Preparation of 3-(prop-1-en-2-yl)cyclohexan-1-one

Copper iodide (11.4 g, 60.0 mmol, 0.6 equiv) was slurried in 750 mL drytetrahydrofuran and chilled in a dry ice-acetone bath. Compound II(0.48M in tetrahydrofuran, 250 mL, 120 mmol, 1.2 equiv) was addeddropwise over the course of one hour to the copper iodide slurry whilethe temperature was kept below −70° C. during the addition.Cyclohexen-1-one (9.6 g, 100.0 mmol, 1.0 equiv) andchlorotrimethylsilane (15.2 mL, 120.0 mmol, 1.2 equiv) were dissolved in50 mL dry tetrahydrofuran. The resulting solution was added dropwiseover the course of 45 minutes to the chilled alkyl copper slurry. Themixture was stirred for an additional fifteen minutes at which time TLC(30% ethyl acetate/hexane) revealed a mixture of desired product, someunreacted cyclohexen-1-one, and two minor side products. The reactionwas quenched with 250 mL of saturated ammonium chloride solution. Thecold bath was then removed and the slurry was allowed to warm to approx.5 C and was poured into a mixture of 50 mL 30% ammonium hydroxidedissolved in 2.5 L of water. The mixture was extracted with 3×1 L ethylacetate and the aqueous phase discarded. The combined organic phaseswere washed with 2×1 L brine then the aqueous phases were discarded. Theorganic phase was concentrated to approx. 200 mL under reduced pressure,diluted to approx. 1 L with diethyl ether then dried over sodiumsulfate. The solids were removed by filtration and discarded and themother liquor was concentrated to obtain an orange oil that was purifiedusing a CombiFlash (330 g column, 0-10% ethyl acetate/hexane) to obtainCompound III as a pale yellow oil. Yield: 6.6 g (48% from II).

Step 3—Preparation of cis-3-(prop-1-en-2-yl)cyclohexan-1-ol IV

Compound III (11.8 g, 85.4 mmol, 1.0 equiv.) was dissolved in 300 mL oftetrahydrofuran. Sodium borohydride (9.7 g, 256.1 mmol, 3.0 equiv) wasadded and the resulting mixture was stirred at room temperature and thereaction was determined to be complete after two hours by TLC (30% ethylacetate/hexane). The reaction was quenched with a minimal amount of 3Maqueous hydrochloric acid until the pH was approx. 2. The solids werefiltered and discarded and the mother liquor was concentrated to an oilusing a rotary evaporator (25 mm Hg, bath temperature 20° C.). The oilwas diluted with 500 mL diethyl ether and dried over sodium sulfate: thesolids were removed by filtration and discarded and the mother liquorwas concentrated to obtain a yellow oil. The material was combined witha previous batch (14 g scale) and purified using a CombiFlash (330 gcolumn, 0-20% ethyl acetate/hexane) to obtain Compound IV as a paleyellow oil. Yield: 13.6 g cis isomer and 1.5 g trans isomer (58% fromIII).

Step 4—Preparation of Compound trans-3-(prop-1-en-2-yl)cyclohexyl4-nitrobenzoate V

Note: this procedure was carried out with 9.5 g of Compound IV, splitinto two 4.75 g batches that were run in parallel.

Compound IV (4.75 g, 33.9 mmol, 1.0 equiv.), was dissolved in 250 mLtetrahydrofuran, followed by 4-nitrobenzoic acid (22.6 g, 135.5 mmol,4.0 equiv) and triphenylphosphine (35.5 g, 135.5 mmol, 4.0 equiv). Theresulting solution was chilled in an ice water bath, after which N,N′-diisopropylazodicarboxylate (26.7 mL, 135.5 mmol, 4.0 equiv) wasadded in small portions over a one hour period, taking care to keep thetemperature below 10° C. during the addition. The ice bath was removedand the reaction was stirred for fifteen hours at room temperature, thenat 40° C. for three hours. After cooling to room temperature, thereaction was diluted with 250 mL diethyl ether and extracted with 2×150mL saturated sodium bicarbonate solution. The combined aqueous phaseswere back extracted with 150 mL diethyl ether: the aqueous phase wasdiscarded and the combined organics were dried over sodium sulfate andconcentrated to obtain a yellow semisolid. At this point the materialfrom both batches was combined and was suspended in 100 mL diethyl etherand allowed to stand overnight. The resulting slurry was diluted with 50mL diethyl ether and 150 mL hexane, and the solids were removed byfiltration and discarded. The mother liquor was concentrated to obtainyellow oil that was split into three portions and purified by CombiFlash(330 g column, 0-15% ethyl acetate/hexane) to obtain 17.4 g pale yellowsolid (Compound V). Yield: 89%.

Step 5—Preparation of Compound trans-3-(prop-1-en-2-yl)cyclohexan-1-olVI

Compound V (17.4 g, 60.1 mmol, 1.0 equiv.) was dissolved in 200 mL oftetrahydrofuran. A solution of lithium hydroxide (8.7 g, dissolved in125 mL of water) was added, and the resulting solution was stirred atroom temperature. The mixture was stirred for fifteen hours anddetermined to be complete by TLC. The reaction mixture was partitionedbetween ether and water and the aqueous phase extracted twice withether. The combined organics were dried over sodium sulfate andconcentrated under reduced pressure to afford a yellow oil, which wasdissolved in 50 mL of dichloromethane and filtered through a 1 cm deepsilica gel pad. The pad was rinsed with a little additionaldichloromethane, and the combined organics were concentrated to drynessto obtain a pale yellow oil that was purified by CombiFlash (120 gcolumn, 0-20% ethyl acetate/hexane, 1 hr) to obtain 6.7 g Compound VI asa colorless oil. Yield: (79%).

Step 6—Preparation of cis-1-(3-tolyloxy)-3-isopropenyl-cyclohexane VII

Compound IV (4.75 g, 33.9 mmol, 1.0 equiv), was dissolved in 250 mLtetrahydrofuran, followed by m-cresol (14.2 mL, 135.5 mmol, 4.0 equiv)and 35.5 g triphenylphosphine (135.5 mmol, 4.0 equiv). The resultingsolution was chilled in an ice water bath, and then N,N′-diisopropylazodicarboxylate (26.7 mL, 135.5 mmol, 4.0 equiv) wasadded in small portions over a one hour period, taking care to keep thetemperature below 10° C. during the addition. The ice bath was removedand the reaction was stirred for fifteen hours at room temperature, thenat 40° C. for three hours. After cooling to room temperature, thereaction was diluted with 250 mL diethyl ether and extracted with 2×150mL saturated sodium bicarbonate solution. The combined aqueous phaseswere back extracted with 150 mL diethyl ether: the aqueous phase wasdiscarded and the combined organics were dried over sodium sulfate. Thesolids were filtered and discarded, and the mother liquor wasconcentrated to obtain orange oil, which was dissolved in 150 mL of 1:1diethyl ether/hexane stirred at room temperature for one hour. Thesolids were removed by filtration and discarded. The mother liquor wasconcentrated to obtain an orange oil that was purified by CombiFlash(330 g column, 0-10% ethyl acetate/hexane) to obtain 5.7 g pale oil(Compound VII). Yield: 73%.

Step 7—Preparation ofcis-1-(3-methylphenyl)-3-(1-hydroxyprop-2-yl)cyclohexane VIII

Note: this procedure was carried out with 5.7 g of Compound VII, splitinto three 1.9 g batches that were run in parallel.

Compound VII (1.9 g, 8.2 mmol, 1.0 eq.) was dissolved in 8.5 mL of drytetrahydrofuran. The solution was chilled in an ice water bath, and thenborane-dimethyl sulfide complex ([2.0M] in tetrahydrofuran, 4.1 mL, 8.2mmol, 1.0 equiv) was added dropwise over a 25 minute period. Theresulting mixture was stirred in the ice water bath for three hours,then aqueous sodium hydroxide solution (3M, 3.3 mL, 9.9 mmol, 1.2 equiv)was added dropwise over a fifteen minute period. Aqueous hydrogenperoxide (35 wt. %, 2.4 mL, 29.5 mmol, 3.6 equiv) was added over a fiveminute period, then the ice bath was removed and the resulting mixturewas stirred at room temperature. TLC (20% ethyl acetate/hexane) infifteen minutes showed starting material was consumed. The reaction wasquenched with 1M aqueous hydrochloric acid, extracted with 3×25 mLdiethyl ether. The combine organics were dried over sodium sulfate: thesolids were removed by filtration and discarded; the mother liquor wasconcentrated to oil and purified by CombiFlash (80 g column, 0-30% ethylacetate/hexane) to obtain 5.4 g of a clear oil (Compound VIII). Yield:75%.

Step 8—Preparation of cis-2-(-3-(m-tolyloxy)cyclohexyl)propanal IX

Compound VIII (50 mg, 0.2 mmol, and 1.0 equiv) was dissolved in 0.75 mLdichloromethane: the resulting solution was chilled in an ice water bathand Dess-Martin periodinane (111 mg, 0.26 mmol, 1.3 equiv) was added andthe mixture stirred in the ice water bath for two hours. TLC (20% ethylacetate/hexane) showed reaction nearly complete. The mixture was stirredat room temperature for 30 minutes, quenched with 1 mL sodiumthiosulfate solution and then extracted with 2×2 mL ethyl acetate. Thecombined organics were extracted with 2 mL of saturated sodiumbicarbonate: the phases were separated and the aqueous phase was backextracted with 4 mL of ethyl acetate. The combined organics were driedover sodium sulfate and the solids were removed by filtration anddiscarded. The mother liquor was concentrated on a rotary evaporator toobtain 50 mg of a yellow oil (Compound IX). The crude material was usedwithout further purification in the next step (Example 9).

Step 8a. Preparation of cis-2-(3-(3-(methoxy)phenoxy)cyclohexyl)propanal

Using the same procedures as described in Steps 6, 7 and 8, but with thesubstitution of m-methoxyphenol in place of m-cresol in Step 6,cis-2-(3-(3-(methoxy)phenoxy)cyclohexyl)propanal was prepared.

Step 8b Preparation ofcis-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propanal

Using the same procedure as described in Steps 6, 7, and 8, but with thesubstitution of m-trifluoromethoxyphenol in place of m-cresol in Step 6,cis-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propanal was prepared.

Example 9a—Preparation ofcis-(N-(pyridin-4-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine)

Cis-2-(-3-(m-tolyloxy)cyclohexyl)propanal (82 mg, 0.33 mmol) wasdissolved in anhydrous THE (3 mL). To this solution was added4-(aminomethyl)pyridine (51 μL, 0.5 mmol) followed by titaniumtetraisopropoxide (202 μL, 0.67 mmol). The mixture was stirred at roomtemperature for 45 min and then cooled in an ice bath. Sodiumtriacetoxyborohydride (212 mg, 1 mmol) was added and the ice bath wasremoved. After 45 minutes at room temperature, LC/MS analysis showed thereaction to be complete. The reaction was quenched with 1 M HCl and mostof the solvent was removed under reduced pressure. The residue wasdissolved in DMSO (1 mL) and purified by HPLC to afford the titleproduct (LC/MS=339.2 [M+H]⁺).

Example 9b—Alternate Preparation ofcis-(N-(pyridin-4-ylmethyl)-2-(3-(m-tolyloxy) cyclohexyl)propan-1-amine)

Cis-2-(-3-(m-tolyloxy)cyclohexyl)propanal (82 mg, 0.33 mmol) wasdissolved in anhydrous dichloroethane (3 mL). To this solution was added4-(aminomethyl)pyridine (51 μL, 0.5 mmole) and the mixture was stirredat room temperature for 45 min after which sodium triacetoxyborohydride(212 mg, 1 mmol) was added and reaction mixture brought to reflux. After45 minutes, LC/MS analysis showed the reaction to be complete. Thereaction was quenched with 1 M HCl and most of the solvent was removedunder reduced pressure. The residue was dissolved in DMSO (1 mL) andpurified by HPLC to afford the title product (LC/MS=339.2 [M+H]⁺).

Example 10—Preparation of cis-N-(4-methylbenzyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine

Using the same procedure described in Example 9, step 2 usingp-tolylmethanamine in place of 4-(aminomethyl)pyridine,cis-N-(4-methylbenzyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=352.2 [M+H]⁺).

Example 11—Preparation ofcis-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl)pyridin-4-amine

Using the same procedure described in Example 9, using pyridin-4-aminein place of 4-(aminomethyl)pyridine,cis-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl)pyridin-4-amine was preparedanalogously (LC/MS=325.2 [M+H]⁺).

Example 12—Preparation ofcis-N-(cyclohexylmethyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, usingcyclohexylmethanamine in place of 4-(aminomethyl)pyridine,cis-N-(cyclohexylmethyl)-2-(3-(m-tolyloxy)-cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=344.2 [M+H]⁺).

Example 13—Preparation ofcis-N-(pyridin-2-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, usingpyridin-2-ylmethanamine in place of 4-(aminomethyl)pyridine,cis-N-(pyridin-2-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-aminewas prepared analogously (LC/MS=339.2 [M+H]⁺).

Example 14—Preparation ofcis-N-(3-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, using (3-chlorophenyl)methanamine in place of 4-(aminomethyl)pyridine,cis-N-(3-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=372.2 [M+H]⁺).

Example 15—Preparation ofcis-2-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino)methyl) aniline

Using the same procedure described in Example 9, using2-(aminomethyl)aniline in place of 4-(aminomethyl)pyridine,cis-N-(3-chlorobenzyl)-2-(3-(m-tolyloxy) cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=353.2 [M+H]⁺).

Example 16—Preparation ofcis-N-(pyridin-3-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, usingpyridin-3-ylmethanamine in place of 4-(aminomethyl)pyridine,cis-N-(pyridin-3-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-aminewas prepared analogously (LC/MS=339.2 [M+H]⁺).

Example 17—Preparation ofcis-N-(2-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, using (2-chlorophenyl)methanamine in place of 4-(aminomethyl)pyridine,cis-N-(2-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=372.2 [M+H]⁺).

Example 18—Preparation ofcis-3-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)aniline

Using the same procedure described in Example 9, using3-(aminomethyl)aniline in place of 4-(aminomethyl)pyridine,cis-3-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)-amino)methyl)aniline wasprepared analogously (LC/MS=353.2 [M+H]⁺).

Example 19—Preparation ofcis-N-(4-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, using (4-chlorophenyl)methanamine in place of 4-(aminomethyl)pyridine,cis-N-(4-chlorobenzyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine wasprepared analogously (LC/MS=371.2[M+H]⁺).

Example 20—Preparation ofcis-N-(4-chlorobenzyl)-N-methyl-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine

Using the same procedure described in Example 9, using1-(4-chlorophenyl)-N-methylmethanamine in place of4-(aminomethyl)pyridine,cis-N-(4-chlorobenzyl)-N-methyl-2-(3-(m-tolyloxy)cyclohexyl)propan-1-aminewas prepared analogously (LC/MS=386.2 [M+H]⁺).

Example 21—Preparation ofcis-2-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)phenol

Using the same procedure described in Example 9, using2-(aminomethyl)phenol in place of 4-(aminomethyl)pyridine,cis-2-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)-amino)methyl)phenol wasprepared analogously (LC/MS=354.2 [M+H]⁺).

Example 22—Preparation ofcis-4-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)aniline

Using the same procedure described in Example 9, using4-(aminomethyl)aniline in place of 4-(aminomethyl)pyridine,cis-4-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)-amino)methyl)aniline wasprepared analogously (LC/MS=353.2 [M+H]⁺).

Example 23—Preparation ofcis-3-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)phenol

Using the same procedure described in Example 9, using3-(aminomethyl)phenol in place of 4-(aminomethyl)pyridine,cis-3-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)phenol wasprepared analogously (LC/MS=354.2 [M+H]⁺).

Example 24—Preparation ofcis-4-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino)methyl) phenol

Using the same procedure described in Example 9, using4-(aminomethyl)phenol in place of 4-(aminomethyl)pyridine,cis-4-(((2-(3-(m-tolyloxy)cyclohexyl)propyl)amino) methyl)phenol wasprepared analogously (LC/MS=354.2 [M+H]⁺).

Example 25—Preparation ofcis-N-(pyrimidin-4-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine

Using the same procedure described in Example 9, usingpyrimidin-4-ylmethanamine in place of 4-(aminomethyl)pyridine,cis-N-(pyrimidin-4-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-aminewas prepared analogously (LC/MS=340.2 [M+H]⁺).

Example 26—Preparation ofcis-N-(pyridin-4-ylmethyl)-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl)acetamide

cis-(N-(pyridin-4-ylmethyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine)(0.338g, 1 mmol), prepared as in Example 9, was dissolved in dichloromethane(5 mL) and treated with acetyl chloride (0.117 g, 1.5 mmol) andtriethylamine (0.152 g, 1.5 mmol), and stirred at 0° C. to roomtemperature for 3 h. The mixture was partitioned between ethyl acetateand sodium bicarbonate solution, and the organic layer was separated,dried and evaporated to dryness to afford the crude product.Purification by preparative HPLC afforded the purecis-N-(pyridin-4-ylmethyl)-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl)acetamide(LC/MS=381.2 [M+H]⁺).

Example 27—Preparation of cis-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine

Using the same procedure described in Example 9, using 7N NH₄OH inmethanol in place of 4-(aminomethyl)pyridine,cis-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine was prepared analogously(LC/MS=248.2 [M+H]⁺).

Example 28—Preparation ofcis-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl)isonicotinamide

2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine (prepared in Example 27) wascoupled with iso-nicotinic acid using a standard coupling procedure(HATU/diisopropylethylamine/DMF) to affordcis-N-(2-(3-(m-tolyloxy)cyclohexyl)propyl) isonicotinamide afterpurification by preparative HPLC (LC/MS=353.2 [M+H]⁺).

Example 29—Preparation ofcis-N-((1H-benzo[d]imidazol-5-yl)methyl)-2-(3-(m-tolyloxy)cyclohexyl)propan-1-amine

Using the procedure described in Example 9a,cis-2-(3-(m-tolyloxy)cyclohexyl) propan-1-amine, prepared in Example 27,was reacted with 1H-benzo[d]imidazole-5-carbaldehyde and purified bypreparative HPLC to affordcis-N-((1H-benzo[d]imidazol-5-yl)methyl)-2-(3-(m-tolyloxy)cyclohexyl)¬propan-1-amine(LC/MS=378.2 [M+H]⁺).

Example 30—Preparation ofcis-N-benzyl-2-(3-(3-methoxyphenoxy)cyclohexyl)propan-1-amine

Using the procedure described in Example 9, substitutingcis-2-(3-(3-(methoxy)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl)propanal, reaction with benzylamineaffordedcis-2-(3-(3-methoxyphenoxy)cyclohexyl)-N-(pyridin-3-ylmethyl)propan-1-amine(LC/MS=354.2 [M+H]⁺).

Example 31—Preparation ofcis-2-(3-(3-methoxyphenoxy)cyclohexyl)-N-(pyridin-3-ylmethyl)propan-1-amine

Using the procedure described in Example 16, substitutingcis-2-(3-(3-(methoxy)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl)propanal, reaction with3-(aminomethyl)pyridine afforded cis-2-(3-(3-methoxyphenoxy)cyclohexyl)-N-(pyridin-3-ylmethyl)propan-1-amine (LC/MS=355.2 [M+H]⁺).

Example 32—Preparation ofcis-2-(3-(3-methoxyphenoxy)cyclohexyl)-N-(pyridin-4-ylmethyl)propan-1-amine

Using the procedure described in Example 9, substitutingcis-2-(3-(3-(methoxy)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl)propanal, reaction with4-(aminomethyl)pyridine afforded cis-2-(3-(3-methoxyphenoxy)cyclohexyl)-N-(pyridin-4-ylmethyl)propan-1-amine (LC/MS=355.2 [M+H]⁺).

Example 33—Preparation of cis-N-benzyl-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine

Using the procedure described in Example 9, substitutingcis-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl) propanal, reaction with benzylamineafforded cis-N-benzyl-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine (LC/MS=392.2 [M+H]⁺).

Example 34—Preparation ofcis-N-(pyridin-3-ylmethyl)-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine

Using the procedure described in Example 16, substitutingcis-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl)propanal, reaction with3-(aminomethyl)pyridine affordedcis-N-(pyridin-3-ylmethyl)-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine(LC/MS=393.2[M+H]⁺).

Example 35—Preparation ofcis-N-(pyridin-4-ylmethyl)-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine

Using the procedure described in Example 9, substitutingcis-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propanal forcis-2-(-3-(m-tolyloxy)cyclohexyl) propanal, reaction with4-(aminomethyl)pyridine affordedcis-N-(pyridin-4-ylmethyl)-2-(3-(3-(trifluoromethyl)phenoxy)cyclohexyl)propan-1-amine(LC/MS=393.2 [M+H]⁺).

Example 36—Screening of C458 Analogs Against Aβ Toxicity in MC65 Cells

The C458-like compounds (TABLE 3) were evaluated using Aβ-producing MC65cells. Several C458 analogs (C594, C596, C600-C603, C607, and C608)demonstrated efficacy against Aβ toxicity comparable to that of CP2 andC458 (FIGS. 11A and 11B).

Example 37—Molecular Mechanism of Action

Using in vitro and in vivo assays, as well as computational chemistry,the molecular mechanism for the action of CP2, C458, and C458 analogswas tested. These studies showed that the first enzymatic complex of themitochondrial oxidative phosphorylation (OXPHOS) machinery that producescellular energy in the form of ATP (Complex I) is a target for new CP2and C458 analogs.

Since CP2 targets Complex I, experiments were conducted to determinewhether the CP2 and C458 analogs also can target Complex I and affectmitochondrial functions such as ATP production, Complex I activity, andNAD⁺/NADH ratios. ATP production was measured in primary corticalneurons treated with CP2 and four novel CP2 analogs (FIGS. 12A and 12B).Compounds C455, C458 and CP2 modestly reduced ATP production over aconcentration range from 10 nM to 5 μM, while C460 and C472 had littleor no effect on ATP production. In good agreement, compounds C455, C458,and CP2 also resulted in 20 to 30 percent inhibition of Complex Iactivity (FIG. 13).

Further, CP2 and C458 analogs, but not an inactive CP2 analog (TP17) oran inactive C458 analog (C599), increased levels of NADH and decreasedthe NAD+/NADH ratio in primary neurons (Zhang et al., eBioMedicine,2015). These studies indicated that active compounds targets Complex Iactivity (FIGS. 14A and 14B), resulting in mild modulation of itsactivity.

Example 38—Screening of Additional C458 Analogs Against Aβ Toxicity inMC65 Cells

The activity of nine (9) new compounds (C687-C695) was tested in an Aβtoxicity assay in MC65 cells (FIGS. 15A and 15B). The structures ofseveral of these compounds were as follows:

All of the new compounds, excluding compound C692, demonstrated a lackof toxicity and high efficacy against Aβ toxicity in these cells.Compounds C693-C695 demonstrated much higher efficacy against Aβtoxicity compared to the rest of the compounds (circled in FIG. 15B).

Example 39—Screening of Additional C458 Analogs for Complex I Inhibitionand Production of ATP, NAD+, and NADH

To further characterize these nine new compounds, Complex I activity wasmeasured in isolated mitochondria, and levels of ATP, NAD+, and NADHwere measured in primary mouse embryonic cortical neurons (FIGS. 16-18).All compounds decreased Complex I activity by 10-25% at 5 μM (FIG. 16).In agreement with the proposed molecular target and the mechanism, allcompounds also mildly inhibited ATP production by 15-25% (FIG. 17).Finally, while all of the new compounds increased levels of NADH anddecreased the NAD+/NADH ratio (FIGS. 18A-18C), C693 and C694demonstrated the highest efficacy.

Example 40—Anti-Cancer Activity of Compounds that Modulate MitochondrialFunction

The effect of CP2 and related compounds on cancer was tested in vitroand in vivo. First, CP2 and C458 were tested in WT mice fed a high fatdiet (FD). Two groups of female mice 288 days old (n=10 mice per group)were used; one group was given CP2 (25 mg/kg via drinking water)starting at 288 days of age (FIG. 19). At 320 days of age, CP2-treatedand untreated mice were put on HFD (60%) for 94 days. After that, bothgroups were on 42% HFD for 272 days (until the age of 686 days). Withinthis period of time, most of the untreated WT mice developed differenttypes of tumors (breast, liver, and abdominal tumors), and 90% of theuntreated WT mice died. In contrast, the CP2-treated mice did notdevelop tumors, and demonstrated increased rates of survival (FIG. 19).Thus, these experiments suggested that CP2 may prevent the developmentof cancer.

To further test this hypothesis, various cancer cell lines were treatedwith different doses (5 μM or 50 μM) of CP2 or compounds C455, C460,C472, or C594, and cell death was evaluated using an MTT assay. Thefollowing human cell lines were used: HeLa (cervical cancer, FIG. 20A);Panc1 (pancreatic cancer, FIG. 20B); P-WT MEFs (control, FIG. 20C), P++MEFs (accelerated aging phenotype, FIG. 20D) p53−/− ms TCL (FIG. 20E);Patu (pancreatic cancer, FIG. 20F), and Su (pancreatic cancer, FIG.20G). The compounds had varying effects on the different cell lines, butin general they suppressed growth of the cancer cell linestested—particularly at the higher dose. The compounds did not affect thesurvival of WT mouse embryonic fibroblasts (FIG. 20C), but they didsuppress the growth of MEFs that were P++(FIG. 20D).

Additional dose response studies were conducted in the same cell linesusing CP2; the results are plotted in FIGS. 21A-21H as follows: FIG.21A, HeLa cervical cancer cells; FIG. 21B, Panc1 pancreatic cancercells; FIG. 21C, P-WT: MEFs; control); FIG. 21D, P++: MEFs; FIG. 21E,p53−/− ms TCL; FIG. 21F, Patu pancreatic cancer cells; FIG. 21G, Supancreatic cancer cells; FIG. 21H, HPDE control.

Taken together, the data obtained in vivo in WT mice (HFD) and in vitrodemonstrated that CP2 and related compounds can prevent the developmentof tumors and suppress the survival of multiple cancer cell lines.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating a subject having apancreatic cancer, cervical cancer, or lymphoma, comprisingadministering to the subject a therapeutically effective amount of acompound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: X is absent orselected from the group consisting of CH₂ and C(O); R¹ is selected fromthe group consisting of H, OH, CN, NO₂, halo, C₁₋₃ alkyl, C₁₋₃haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇ cycloalkyl, amino, C₁₋₃alkylamino, and di(C₁₋₃ alkyl)amino; R² is selected from the groupconsisting of H and C₁₋₆ alkyl; R³ is selected from the group consistingof H, C₁₋₆ alkyl, —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl); R⁴ isselected from the group consisting of C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocycloalkyl, each optionallysubstituted by 1, 2, 3, or 4 independently selected R⁵ groups; and R⁵ isselected from the group consisting of OH, CN, NO₂, halo, C₁₋₃ alkyl,C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇ cycloalkyl, amino,C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.
 2. The method of claim 1,wherein the subject is a human.
 3. The method of claim 1, wherein X isCH₂.
 4. The method of claim 3, wherein R¹ is C₁₋₃ alkyl.
 5. The methodof claim 4, wherein R² is C₁₋₆ alkyl.
 6. The method of claim 5, whereinR³ is H.
 7. The method of claim 6, wherein R⁴ is unsubstituted 5-10membered heteroaryl.
 8. The method of claim 6, wherein R⁴ is aryl and R⁵is halo or OH.
 9. The method of claim 1, wherein the compound of Formula(I) is a compound of Formula (Ia):

or a pharmaceutically acceptable salt thereof.
 10. The method of claim1, wherein the compound of Formula (I) is a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is selectedfrom the group consisting of H, OH, CN, NO₂, halo, C₁₋₃ alkyl, C₁₋₃haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇ cycloalkyl, amino, C₁₋₃alkylamino, and di(C₁₋₃ alkyl)amino; R² is selected from the groupconsisting of H and C₁₋₆ alkyl; R³ is selected from the group consistingof H, C₁₋₆ alkyl, —C(O)(C₁₋₃ alkyl), and —C(O)O(C₁₋₃ alkyl); R⁴ isselected from the group consisting of C₃₋₁₀ cycloalkyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocycloalkyl, each optionallysubstituted by 1, 2, 3, or 4 independently selected R⁵ groups; and R⁵ isselected from the group consisting of OH, CN, NO₂, halo, C₁₋₃ alkyl,C₁₋₃ haloalkyl, C₁₋₃ alkoxy, C₁₋₃ haloalkoxy, C₃₋₇ cycloalkyl, amino,C₁₋₃ alkylamino, and di(C₁₋₃ alkyl)amino.
 11. The method of claim 10,wherein R¹ is C₁₋₃ alkyl.
 12. The method of claim 11, wherein R² is C₁₋₆alkyl.
 13. The method of claim 12, wherein R³ is H.
 14. The method ofclaim 13, wherein R⁴ is unsubstituted 5-10 membered heteroaryl.
 15. Themethod of claim 13, wherein R⁴ is aryl and R⁵ is halo or OH.
 16. Themethod of claim 10, wherein the compound of Formula (II) is a compoundof Formula (IIa)

or a pharmaceutically acceptable salt thereof.
 17. The method of claim1, wherein the compound of Formula (I) is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.